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Research article

Vol 20 No 1 (2026): Volume 20, Number 1, 2026

Optimization of energy performance in a pilot-scale biofilm anaerobic reactor through feedstock composition control of limonene-containing fruit waste

DOI
https://doi.org/10.22146/jrekpros.26702
Submitted
December 3, 2025
Published
June 9, 2026

Abstract

Fruit waste containing citrus fractions with high limonene content presents a particular challenge for anaerobic digestion (AD) due to the inhibitory effects of this terpene on methanogenic microorganisms. This study evaluates the performance of a pilot-scale biofilm anaerobic reactor treating mixed fruit waste with varying proportions of citrus residues, focusing on biogas production, methane composition, and process stability. A 1,167 L fiberglass reactor equipped with K1 biofilm carriers was operated under mesophilic conditions and fed with six different substrate formulations. The results indicate that increasing the proportion of citrus waste leads to reduced biogas production during the initial phase, accompanied by volatile fatty acid (VFA) accumulation and pH suppression, suggesting process inhibition driven by limonene. In contrast, formulations with moderated citrus content showed improved operational stability, characterized by pH values of 6.8–7.1, VFA/alkalinity ratios below 0.30, and higher methane fractions. Energy conversion analysis revealed that treatments with controlled limonene levels yielded the highest net energy output, while excessive citrus content decreased conversion efficiency. Correlation analysis confirmed strong associations between VFA accumulation, pH decline, and reduced methane concentration under high-limonene conditions. These findings demonstrate that managing the proportion of citrus-rich fruit waste is essential for maintaining process stability and enhancing biogas productivity in pilot-scale biofilm AD systems.

References

  1. Agrawal, A., Chaudhari, P. K., & Ghosh, P. (2023). Anaerobic digestion of fruit and vegetable waste: a critical review of associated challenges. In Environmental Science and Pollution Research (Vol. 30, Issue 10, pp. 24987–25012). Springer Science and Business Media Deutschland GmbH. https://doi.org/10.1007/s11356-022-21643-7
  2. Ali, H. A., Faraj, J. J., & Hussien, F. M. (2021). Effect of pH on biogas production during anaerobic digestion.
  3. Ali, M., Pena, C. B., & De Vrieze, J. (2025). Anaerobic co-digestion of sewage sludge and food waste: staging and carriers enhance system performance and process stability. Bioresource Technology Reports, 102329. https://doi.org/10.1016/j.biteb.2025.102329
  4. Alvarado-Vallejo, A., Marín-Peña, O., Rosas-Mendoza, E. S., Méndez-Contreras, J. M., & Alvarado-Lassman, A. (2024). The Valorization of Fruit and Vegetable Wastes Using an Anaerobic Fixed Biofilm Reactor: A Case of Discarded Tomatoes from a Traditional Market. Processes, 12(9). https://doi.org/10.3390/pr12091923
  5. Al-Zoubi, A. I., Alkhamis, T. M., & Alzoubi, H. A. (2024). Optimized biogas production from poultry manure with respect to pH, C/N, and temperature. Results in Engineering, 22. https://doi.org/10.1016/j.rineng.2024.102040
  6. Appels, L., Baeyens, J., Degrève, J., & Dewil, R. (2008). Principles and potential of the anaerobic digestion of waste-activated sludge. In Progress in Energy and Combustion Science (Vol. 34, Issue 6, pp. 755–781). https://doi.org/10.1016/j.pecs.2008.06.002
  7. Atasoy, M., & Cetecioglu, Z. (2022). The effects of pH on the production of volatile fatty acids and microbial dynamics in long-term reactor operation. Journal of Environmental Management, 319. https://doi.org/10.1016/j.jenvman.2022.115700
  8. Azevedo, A., Lapa, N., Moldão, M., Gominho, J., & Duarte, E. (2025). Fruit and vegetable wastes as co-substrates in anaerobic co-digestion: Effect of storage temperature on physicochemical properties and biogas production. Energy Nexus, 17. https://doi.org/10.1016/j.nexus.2024.100354
  9. Banarase, S. E., & Phirke, N. V. (2024). Enhancing biogas production from vegetables and fruits wastes by applying effective strategies. Ecology, Environment and Conservation, 30(02), 930–935. https://doi.org/10.53550/eec.2024.v30i02.084
  10. Battista, F., Remelli, G., Zanzoni, S., & Bolzonella, D. (2020). Valorization of Residual Orange Peels: Limonene Recovery, Volatile Fatty Acids, and Biogas Production. ACS Sustainable Chemistry and Engineering, 8(17), 6834–6843. https://doi.org/10.1021/acssuschemeng.0c01735
  11. Beschkov, V. N., & Angelov, I. K. (2025). Volatile Fatty Acid Production vs. Methane and Hydrogen in Anaerobic Digestion. In Fermentation (Vol. 11, Issue 4). Multidisciplinary Digital Publishing Institute (MDPI). https://doi.org/10.3390/fermentation11040172
  12. Bouallagui, H., Haouari, O., Touhami, Y., Ben Cheikh, R., Marouani, L., & Hamdi, M. (2004). Effect of temperature on the performance of an anaerobic tubular reactor treating fruit and vegetable waste. Process Biochemistry, 39(12), 2143–2148. https://doi.org/10.1016/j.procbio.2003.11.022
  13. Budiyono, Manthia, F., Amalin, N., Hawali Abdul Matin, H., & Sumardiono, S. (2018). Production of Biogas from Organic Fruit Waste in Anaerobic Digester using Ruminant as the Inoculum. MATEC Web of Conferences, 156. https://doi.org/10.1051/matecconf/201815603053
  14. Chen, Y., Cheng, J. J., & Creamer, K. S. (2008). Inhibition of anaerobic digestion process: A review. In Bioresource Technology (Vol. 99, Issue 10, pp. 4044–4064). https://doi.org/10.1016/j.biortech.2007.01.057
  15. Chin, M. J., Poh, P. E., Tey, B. T., Chan, E. S., & Chin, K. L. (2013). Biogas from palm oil mill effluent (POME): Opportunities and challenges from Malaysia’s perspective. In Renewable and Sustainable Energy Reviews (Vol. 26, pp. 717–726). https://doi.org/10.1016/j.rser.2013.06.008
  16. Chubur, V., Hasan, G., Kára, J., Hanzlíková, I., Chernysh, Y., Sedláček, J., Wang, J., & Roubík, H. (2024). Utilization of citrus, date, and jujube substrates for anaerobic digestion processes. Biofuels, Bioproducts and Biorefining, 18(6), 1917–1929. https://doi.org/10.1002/bbb.2665
  17. Gebresilasie, G. G., Gebreslassie, M. G., & Gebresemati, M. (2025a). Comparative potential of biogas production from the distillery, fruit and vegetable waste and their mixtures (digestion). Heliyon, 11(2). https://doi.org/10.1016/j.heliyon.2025.e42068
  18. Gebresilasie, G. G., Gebreslassie, M. G., & Gebresemati, M. (2025b). Comparative potential of biogas production from the distillery, fruit and vegetable waste and their mixtures (digestion). Heliyon, 11(2). https://doi.org/10.1016/j.heliyon.2025.e42068
  19. Hussein, N., Liew, S. M., Hanafi, N. S., Lee, P. Y., Cheong, A. T., Ghazali, S. S., Chinna, K., Pang, Y. K., Kassim, A., Parker, R. A., Schwarze, J., Sheikh, A., Pinnock, H., & Khoo, E. M. (2023). Asthma control and care among six public health clinic attenders in Malaysia: A cross-sectional study. Health Science Reports, 6(5). https://doi.org/10.1002/hsr2.1021
  20. Jameel, M. K., Mustafa, M. A., Ahmed, H. S., Mohammed, A. jassim, Ghazy, H., Shakir, M. N., Lawas, A. M., Mohammed, S. khudhur, Idan, A. H., Mahmoud, Z. H., Sayadi, H., & Kianfar, E. (2024). Biogas: Production, properties, applications, economic and challenges: A review. In Results in Chemistry (Vol. 7). Elsevier B.V. https://doi.org/10.1016/j.rechem.2024.101549
  21. Li, X., Chu, S., Zhang, Y., Zhou, Y., Su, Y., Wu, D., Hong, S., Zhan, M., & Xie, B. (2026). Impacts of biogas residual biochar on anaerobic digestion of food waste with different organic loading rate: Methanogenic performance and metabolic pathways. Fuel, 407, 137256. https://doi.org/10.1016/j.fuel.2025.137256
  22. Lotito, A. M., De Sanctis, M., Pastore, C., & Di Iaconi, C. (2018). Biomethanization of citrus waste: Effect of waste characteristics and of storage on treatability and evaluation of limonene degradation. Journal of Environmental Management, 215, 366–376. https://doi.org/10.1016/j.jenvman.2018.03.057
  23. Marendra, F., Pramudikto, D. A., Rahmada, A., Rimbawan, H. J., Cahyono, R. B., & Ariyanto, T. (2020). Biogas production for electricity from fruit waste: A case study of Gemah Ripah biogas plant, Yogyakarta. IOP Conference Series: Materials Science and Engineering, 736(2). https://doi.org/10.1088/1757-899X/736/2/022058
  24. Marendra, F., Sarnes, R., Ramadhania Putri Maresi, S., Puspaduhita, A., Dwi Sutarni, Y., & Mustafawi Muhammadi, F. (2025). Pengaruh Kadar Senyawa Kapsaisinoid terhadap Produksi Biogas dalam Proses Anaerobic Digestion Limbah Sayur. Warta Akab, 49(1), 5–14.
  25. Mgeni, S. T., Mero, H. R., Mtashobya, L. A., & Emmanuel, J. K. (2024). The prospect of fruit wastes in bioethanol production: A review. In Heliyon (Vol. 10, Issue 19). Elsevier Ltd. https://doi.org/10.1016/j.heliyon.2024.e38776
  26. Mishra, S., Huang, Y., Li, J., Wu, X., Zhou, Z., Lei, Q., Bhatt, P., & Chen, S. (2022). Biofilm-mediated bioremediation is a powerful tool for the removal of environmental pollutants. In Chemosphere (Vol. 294). Elsevier Ltd. https://doi.org/10.1016/j.chemosphere.2022.133609
  27. Mohmed Moffit, M. A., Suja’, F., Kabir Ahmad, I., & Bhaskaran, A. N. (2025). Biogas production and reactor performance of a pilot scale anaerobic biofilm digester treating food waste. Renewable Energy, 243. https://doi.org/10.1016/j.renene.2025.122414
  28. Nabati, E. G., Nieto, M. T. A., Bode, D., Schindler, T. F., Decker, A., & Thoben, K. D. (2022). Challenges of manufacturing for energy efficiency: towards a systematic approach through applications of machine learning. Production, 32. https://doi.org/10.1590/0103-6513.20210147
  29. Olaimat, A. N., Ababneh, A. M., Al-Holy, M., Al-Nabulsi, A., Osaili, T., Abughoush, M., Ayyash, M., & Holley, R. A. (2024). A Review of Bacterial Biofilm Components and Formation, Detection Methods, and Their Prevention and Control on Food Contact Surfaces. In Microbiology Research (Vol. 15, Issue 4, pp. 1973–1992). Multidisciplinary Digital Publishing Institute (MDPI). https://doi.org/10.3390/microbiolres15040132
  30. Ozadowicz, A., & Grela, J. (2017). Impact of building automation control systems on energy efficiency - university building case study. IEEE International Conference on Emerging Technologies and Factory Automation, ETFA, 1–8. https://doi.org/10.1109/ETFA.2017.8247686
  31. Pambudi, N. A., Firdaus, R. A., Rizkiana, R., Ulfa, D. K., Salsabila, M. S., Suharno, & Sukatiman. (2023). Renewable Energy in Indonesia: Current Status, Potential, and Future Development. In Sustainability (Switzerland) (Vol. 15, Issue 3). MDPI. https://doi.org/10.3390/su15032342
  32. Patinvoh, R. J., Lundin, M., Taherzadeh, M. J., & Sárvári Horváth, I. (2020). Dry Anaerobic Co-Digestion of Citrus Wastes with Keratin and Lignocellulosic Wastes: Batch And Continuous Processes. Waste and Biomass Valorization, 11(2), 423–434. https://doi.org/10.1007/s12649-018-0447-y
  33. Plazzotta, S., Cottes, M., Simeoni, P., & Manzocco, L. (2020). Evaluating the environmental and economic impact of fruit and vegetable waste valorisation: The lettuce waste study-case. Journal of Cleaner Production, 262. https://doi.org/10.1016/j.jclepro.2020.121435
  34. Praharasti, A. S., Febrisiantosa, A., Jatmiko, T. H., Wahono, S. K., Prasetyo, D. J., Pratiwi, D., Suryani, R., Rizal, W. A., Hidayat, N., & Suwanto, A. (2023). Pembuatan pupuk padat dari hasil sampaing biogas di Gunungkidul. Jurnal Penelitian Pertanian Terapan, 23(2), 167–174. https://doi.org/10.25181/jppt.v23i2.2291
  35. Purwasih, R., Sutaryo, S., Purnomoadi, A., Purbowati, E., & Haryanto, B. (2025). Anaerobic digestion from fruit waste for biogas production to sustainable development: A comprehensive bibliometric analysis. Sustainable Futures, 10. https://doi.org/10.1016/j.sftr.2025.101255
  36. Qamar, S., Zhang, Q., Ahmad, M., Pinzón, S., Ahmed, N., & Işık, C. (2025). Roadmap to green future: Biodigester technology acceptance to upscale rural energy access. Energy, 315. https://doi.org/10.1016/j.energy.2025.134437
  37. Rahmada, A., Marendra, F., Rimbawan, H., Wulandari, C., Putri, A. E., Mustofa, A., Pramudito, D. A., Millati, R., Cahyono, R. B., & Ariyanto, T. (2021). Zero Waste Concept in Fruit Waste Anaerobic Digester: Case Study of Biogas Plant Gamping, Yogyakarta. Advances in Engineering Research, 202, 246–251.
  38. Rahmanta, A. P. (2025). Sttetegi operasi biodigester dengan substrat inhibitor limonen dan tanin dari sampah buah. Fruitset Sains, 12(6), 362–372.
  39. Rather, M. A., Gupta, K., & Mandal, M. (2021). Microbial biofilm: formation, architecture, antibiotic resistance, and control strategies. In Brazilian Journal of Microbiology (Vol. 52, Issue 4, pp. 1701–1718). Springer Science and Business Media Deutschland GmbH. https://doi.org/10.1007/s42770-021-00624-x
  40. Renggaman, A., Choi, H. L., Sudiarto, S. I. A., Suresh, A., & Jeon, Y. C. (2024). Biomethane Potential of Beef Cattle Slaughterhouse Waste and the Impact of Co-Digestion with Cattle Feces and Swine Slurry. Fermentation, 10(10). https://doi.org/10.3390/fermentation10100510
  41. Ripley, L. E., & Boyle, W. C. (1986). Improved Alkalimetric Monitoring for Anaerobic Digestion of High-Strength Wastes. In Converse Source: Journal (Water Pollution Control Federation) (Vol. 58, Issue 5).
  42. Rocha, D. H. D., Freitas, F. R. S., Sakamoto, I. K., Silva, E. L., & Varesche, M. B. A. (2024). Co-digestion of solid-liquid waste from citrus agroindustrial: Effect of hydraulic retention time and organic loading rate on H2 production in a long-term continuous operation leach bed reactor. International Journal of Hydrogen Energy, 49, 554–571. https://doi.org/10.1016/j.ijhydene.2023.10.289
  43. Rokaya, B., Kerroum, D., Hayat, Z., Panico, A., Ouafa, A., & Pirozzi, F. (2024). Biogas production by an anaerobic digestion process from orange peel waste and its improvement by limonene leaching: Investigation of H2O2 pre-treatment effect. Energy Sources, Part A: Recovery, Utilization and Environmental Effects, 46(1), 2704–2712. https://doi.org/10.1080/15567036.2019.1692975
  44. Rouhollahi, Z., Ebrahimi-Nik, M., Ebrahimi, S. H., Abbaspour-Fard, M. H., Zeynali, R., & Bayati, M. R. (2020). Farm biogas plants, a sustainable waste to energy and bio-fertilizer opportunity for Iran. Journal of Cleaner Production, 253. https://doi.org/10.1016/j.jclepro.2019.119876
  45. Ruiz, B., & Flotats, X. (2016a). Effect of limonene on batch anaerobic digestion of citrus peel waste. Biochemical Engineering Journal, 109, 9–18. https://doi.org/10.1016/j.bej.2015.12.011
  46. Ruiz, B., & Flotats, X. (2016b). Effect of limonene on batch anaerobic digestion of citrus peel waste. Biochemical Engineering Journal, 109, 9–18. https://doi.org/10.1016/j.bej.2015.12.011
  47. Siegert, I., & Banks, C. (2005). The effect of volatile fatty acid additions on the anaerobic digestion of cellulose and glucose in batch reactors. Process Biochemistry, 40(11), 3412–3418. https://doi.org/10.1016/j.procbio.2005.01.025
  48. Soeprijanto, S., Anfi, R., Fatullah, S., Agustina, D., Firdha, A., & Kaisar, A. A. (2020). Biogas Production from Vegetables and Fruit Wastes Using Anaerobic Floating Bioreactor (Vol. 17, Issue 2).
  49. Suarez, E., Tobajas, M., Mohedano, A. F., & de la Rubia, M. A. (2022). Energy recovery from food waste and garden and park waste: Anaerobic co-digestion versus hydrothermal treatment and anaerobic co-digestion. Chemosphere, 297. https://doi.org/10.1016/j.chemosphere.2022.134223
  50. Sun, Y., Chen, S., Zhang, C., Liu, Y., Ma, L., & Zhang, X. (2018). Effects of sub-minimum inhibitory concentrations of lemon essential oil on the acid tolerance and biofilm formation of Streptococcus mutans. Archives of Oral Biology, 87, 235–241. https://doi.org/10.1016/j.archoralbio.2017.12.028
  51. Uruén, C., Chopo-Escuin, G., Tommassen, J., Mainar-Jaime, R. C., & Arenas, J. (2020). antibiotics Biofilms as Promoters of Bacterial Antibiotic Resistance and Tolerance. https://doi.org/10.3390/antibiotics
  52. Wanasolo, W., Manyele, S. V., & Makunza, J. (2013). A Kinetic Study of Anaerobic Biodegradation of Food and Fruit Residues during Biogas Generation Using Initial Rate Method. Engineering, 05(07), 577–586. https://doi.org/10.4236/eng.2013.57070
  53. Wang, X., Huang, S., Wang, S., Chen, S., Dong, S., & Zhu, Y. (2024). Effect of D-limonene on volatile fatty acidsproduction from anaerobic fermentation of waste activated sludge under pH regulation: Performance and mechanisms. Journal of Environmental Management, 370. https://doi.org/10.1016/j.jenvman.2024.122828
  54. Ward, A. J., Hobbs, P. J., Holliman, P. J., & Jones, D. L. (2008). Optimisation of the anaerobic digestion of agricultural resources. In Bioresource Technology (Vol. 99, Issue 17, pp. 7928–7940). https://doi.org/10.1016/j.biortech.2008.02.044
  55. Wikandari, R., Nguyen, H., Millati, R., Niklasson, C., & Taherzadeh, M. J. (2015). Improvement of biogas production from orange peel waste by leaching of limonene. BioMed Research International, 2015. https://doi.org/10.1155/2015/494182
  56. Wu, K. K., Zhao, L., Wang, Z. H., Sun, Z. F., Wu, J. T., Chen, C., Xing, D. F., Yang, S. S., Wang, A. J., Zhang, Y. F., & Ren, N. Q. (2024). Simultaneous biogas upgrading and medium-chain fatty acids production using a dual membrane biofilm reactor. Water Research, 249. https://doi.org/10.1016/j.watres.2023.120915
  57. Yin, D. min, Mahboubi, A., Wainaina, S., Qiao, W., & Taherzadeh, M. J. (2021). The effect of mono- and multiple fermentation parameters on volatile fatty acids (VFAs) production from chicken manure via anaerobic digestion. Bioresource Technology, 330. https://doi.org/10.1016/j.biortech.2021.124992
  58. Zhang, C., Su, H., Baeyens, J., & Tan, T. (2014). Reviewing the anaerobic digestion of food waste for biogas production. In Renewable and Sustainable Energy Reviews (Vol. 38, pp. 383–392). Elsevier Ltd. https://doi.org/10.1016/j.rser.2014.05.038
  59. Zhou, Y., Zhu, J., Huang, X., Gao, J., & Zhuang, G. (2023). Performance and microbial characteristics of anaerobic/anoxic/aerobic (A2O)-biofilm system for electroplating wastewater treatment: a pilot scale study. Desalination and Water Treatment, 313, 74–81. https://doi.org/10.5004/dwt.2023.30069
  60. Zhu, G. F., Li, J. Z., Wu, P., Jin, H. Z., & Wang, Z. (2008). The performance and phase separated characteristics of an anaerobic baffled reactor treating soybean protein processing wastewater. Bioresource Technology, 99(17), 8027–8033. https://doi.org/10.1016/j.biortech.2008.03.046